title: Use of Kaede fusions to visualize recycling of G protein-coupled receptors.
abstract: The heptahelical G protein-coupled receptors (GPCRs) are internalized following agonist treatment and either recycle rapidly to the plasma membrane or enter the lysosomal degradation pathway. Many conventional GPCR recycling assays suffer from the fact that receptors arriving from the secretory pathway may interfere with recycling receptors. In this study, we introduce a new methodology to study post-endocytotic GPCR trafficking using fusions with the recently cloned Kaede protein. In contrast to the widely used green fluorescent protein, the fluorescence of Kaede can be converted from green to red using ultraviolet irradiation. Our methodology allows to study recycling of GPCRs microscopically in real-time bypassing problems with secretory pathway receptors. Initially, receptors are internalized using an agonist. Fluorescence signals in endosomes are switched, and trafficking of the receptors to the plasma membrane can be easily visualized by monitoring their new fluorescence. Using this methodology, we show that the corticotropin-releasing factor receptor type 1 belongs to the family of recycling GPCRs. Moreover, we demonstrate by fluorescence correlation spectroscopy that Kaede does not oligomerize when fused to membrane proteins, representing an additional advantage of this technique. The Kaede technology may be a powerful tool to study membrane protein trafficking in general.
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, Berlin, Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Schmidt
authors.forename: Burkhard
authors.initials: B
authors.lastname: Wiesner
authors.forename: Klaus
authors.initials: K
authors.lastname: Weisshart
authors.forename: Katharina
authors.initials: K
authors.lastname: Schulz
authors.forename: Jens
authors.initials: J
authors.lastname: Furkert
authors.forename: Björn
authors.initials: B
authors.lastname: Lamprecht
authors.forename: Walter
authors.initials: W
authors.lastname: Rosenthal
authors.forename: Ralf
authors.initials: R
authors.lastname: Schülein
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D008024:Ligands; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D010777:Photochemistry; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
chemical_list: C513582:Kaede protein, Trachyphyllia geoffroyi; D008024:Ligands; D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled